Disposable test strips with integrated reagent/blood separation layer

ABSTRACT

An improved disposable glucose test strip for use in a test meter of the type which receives a disposable test strip and a sample of blood from a patient and performs an electrochemical analysis is made using a non-conductive integrated reagent/blood separation layer containing a filler, an enzyme effective to oxidize glucose, e.g., glucose oxidase, and a mediator effective to transfer electrons from the enzyme. The integrated layer formulation is printed over a conductive carbon element to form a working electrode. The filler, for example a silica filler, is selected to have a balance of hydrophobicity and hydrophilicty such that one drying it forms a two-dimensional network on the surface of the conductive element. The response of this test strip is essentially temperature independent over relevant temperature ranges and is substantially insensitive to the hematocrit of the patient.

BACKGROUND OF THE INVENTION

This application relates to disposable test strips for use inelectrochemical determinations of blood analytes such as glucose, and tomethods and compositions for use in making such strips.

Glucose monitoring is a fact of everyday life for diabetic individuals,and the accuracy of such monitoring can literally mean the differencebetween life and death. To accommodate a normal life style to the needfor frequent monitoring of glucose levels, a number of glucose metersare now available which permit the individual to test the glucose levelin a small amount of blood.

Many of these meters detect glucose in a blood sample electrochemically,by detecting the oxidation of blood glucose using an enzyme such asglucose oxidase provided as part of a disposable, single-use electrodesystem. Examples of devices of this type are disclosed in EuropeanPatent No. 0 127 958, and U.S. Pat. Nos. 5,141,868, 5,286,362,5,288,636, and 5,437,999 which are incorporated herein by reference forpurposes of those countries which permit such incorporation.

In general, existing glucose test strips for use in electrochemicalmeters comprise a substrate, working and reference electrodes formed onthe surface of the substrate, and a means for making connection betweenthe electrodes and the meter. The working electrode is coated with anenzyme capable of oxidizing glucose, and a mediator compound whichtransfers electrons from the enzyme to the electrode resulting in ameasurable current when glucose is present. Representative mediatorcompounds include ferricyanide, metallocene compounds such as ferrocene,quinones, phenazinium salts, redox indicator DCPIP, andimidazole-substituted osmium compounds.

Working electrodes of this type have been formulated in a number ofways. For example, mixtures of conductive carbon, glucose oxidase and amediator have been formulated into a paste or ink and applied to asubstrate. EP 0 127 958 and U.S. Pat. No. 5,286,362. In the case ofdisposable glucose strips, this application is done by screen printingin order to obtain the thin layers suitable for a small flat test strip.The use of screen printing, however, introduces problems to theoperation of the electrode.

Unlike a thicker carbon paste electrode which remains fairly intactduring the measurement, screen printed electrodes formed from carbonpastes or inks are prone to break up on contact with the sample. Theconsequences of this breakup are two-fold. Firstly, the components ofthe electrode formulation are released into solution. Once thesecomponents drift more than a diffusion length away from the underlyingconductive layer, they no longer contribute toward the measurement, butin fact diminish the response by depleting inwardly-diffusing analyte.Secondly, the breakup of the screen printed electrode means that theeffective electrode area is falling over time.

The combination of these two effects results in current transients whichfall rapidly from an initial peak over the period of the measurement,and a high sensitivity to oxygen which quickly competes with themediator for the enzyme. This fact is clearly demonstrated by the muchlower currents measured in blood samples than in plasma samples or otheraqueous media, and can result in erroneous readings. A furtherconsequence is that the transients are often “lumpy” as the electrodebreaks up in a chaotic manner. Lumpy transients either give rise toerroneous readings or rejected strips, neither of which are acceptable.

In addition to the potential for electrode breakup of screen-printedcarbon-based electrodes, known electrodes used in disposable glucosetest strips have been kinetically-controlled, i.e., the current dependson the rate of conversion of glucose by the enzyme. Because the responsemeasured by the instrument represents a balance between the reactions ofenzyme and mediator, enzyme and glucose and enzyme and oxygen, andbecause each of these reactions has its own dependence on temperature,the response of a kinetically-controlled test strip is very sensitive tothe temperature of the sample. Substantial variation in the measuredglucose value can therefore occur as a result of variations in samplehandling.

A further challenge facing sensors for electrochemical glucose detectionarises as a result of interference from blood cells present in thesample. The level of red blood cells is reflected in the hematocritreading. Typically, high hematocrit samples results in readings that arelower than the true value, while low hematocrit samples result inreadings that are higher because the blood cells tend to foul thesurface of the electrode and limit electron transfer. Also, oxygen boundto the hemoglobin of red blood cells competes with the mediator for thereduced enzyme, thereby further diminishing the glucose response.Attempts have been made to limit the hematocrit effect by adding amembrane to filter out blood components (see, U.S. Pat. No. 5,658,444,which is incorporated herein by reference for purposes of thosecountries which permit such incorporation), but this adds an extra stepto the manufacturing process, with associated increase in cost and oftendegraded performance in other areas such as precision.

Because of the importance of obtaining accurate glucose readings to thewell-being of a patient using the meter and disposable test strips, itwould be highly desirable to have a glucose test strip which did notsuffer from these drawbacks, and which therefore provided a moreconsistent and reliable indication of actual blood glucose values,regardless of actual conditions. It is therefore an object of thepresent invention to provide disposable glucose test strips whichprovide a glucose reading that is essentially independent of thehematocrit of the sample, an which include an integrated reagent/bloodseparation layer.

It is further object of the present invention to provide an improvedmethod for making disposable glucose test strips.

SUMMARY OF THE INVENTION

The present invention provides an improved disposable test strip for usein a test meter of the type which receives a disposable test strip and asample of blood from a patient and performs an electrochemical analysisof the amount of a blood analyte such as glucose in the sample. The teststrip comprises:

(a) a substrate;

(b) a first conductive element disposed on the substrate;

(c) a second conductive element disposed on the substrate in sufficientproximity to the first conductive element to allow the completion of anelectrical circuit between the first and second conductive elements whena sample of blood is placed on the test strip;

-   -   (d) a non-conductive integrated reagent/blood separation layer        disposed over the first conductive element; and    -   (e) contacts for making an electrical connection between the        first and second conductive elements and the test meter. The        integrated reagent/blood separation layer comprises reagents for        the electrochemical detection of the analyte dispersed in a        non-conductive matrix effective to exclude blood cells from the        surface of the first conductive element while permitting access        to the first conductive element by soluble electroactive        species. In one embodiment of the invention, a glucose test        strip is formed with an integrated reagent/blood separation        layer comprising a filler which has both hydrophobic and        hydrophilic surface regions, an enzyme effective to oxidize        glucose, e.g., glucose oxidase, and a mediator effective to        transfer electrons from the enzyme to the conductive element.        The filler is selected to have a balance of hydrophobicity and        hydrophilicity such that on drying the integrated reagent/blood        separation layer forms a two-dimensional network on the surface        of the conductive element. Preferred integrated reagent/blood        separation layers comprise non-conductive silica fillers in        combination with materials such as hydroxyethyl cellulose (HEC).        The silica and HEC form a two-dimensional network which excludes        red blood cells, thus rendering the test strip substantially        insensitive to the hematocrit of the patient.

In a preferred embodiment of the invention, the test strips are preparedwith an insulation layer disposed over at least the first conductiveelement. This insulation layer has an aperture formed in it which isaligned with a portion of the first conductive element, and theintegrated reagent/blood separation layer is disposed to make contactwith the first conductive element through this aperture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show an electode structure useful in a disposable teststrip in accordance with the invention;

FIG. 2 shows a test strip in accordance with the invention;

FIGS. 3A-3C show the current measured as a function of glucoseconcentration for three different hematocrit levels;

FIG. 4 shows the relationship of the glucose-concentration dependence ofthe measured current as a function of hematocrit;

FIGS. 5A-5C show the current measured as a function of glucose in bloodand a control solution for three different conductive elements;

FIGS. 6A and 6B show the current measured as a function of glucose attwo different temperatures;

FIG. 7 shows a further embodiment of a glucose test strip according tothe invention;

FIGS. 8A and 8B show current transients observed using a test stripaccording to the invention and a commercial carbon-based test strip;

FIGS. 9A-C show a three-step process for manufacture of test strips inaccordance with the invention; and

FIGS. 10A-10G show the manufacture of a test strip in accordance withthe invention.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 1A and 1B show electrodes useful in a disposable test strip inaccordance with the invention. As shown, the electrodes are formed on asubstrate 10. On the substrate 10 are placed two conductive elements 14′and 16, connected by leads 14 and 15 to conductive contacts 11, 12; and13. An insulating mask 18 is then formed, leaving at least a portion ofconductive elements 14′ and 16, and the contacts 11, 12 and 13 exposed.A non-conductive integrated reagent/blood separation layer 17 is thenapplied over the insulating mask 18 to make contact with conductiveelement 16.

The assembly shown in FIG. 1 provides a fully functional assembly forthe measurement of a blood analyte when connected to a meter.Advantageously, however, the electrode strips of the invention arefinished by applying a nylon or polyester mesh 21 over the sampleapplication region defined by the location of the integratedreagent/blood separation layer 17 of the electrode assembly 22, and thena top cover 23 to prevent splashing of the blood sample. (FIG. 2) Thepolyester mesh acts to guide the sample to the reference electrode,conductive element 14′, thereby triggering the device and initiating thetest.

The utilization of a non-conductive integrated reagent/blood separationlayer provides an important distinction from and advantage over knowntest strips which utilize a conductive reagent-containing slurry toprint the reagents. In these systems, the printed slurry becomes afunctional part of the electrode and charge transfer can take place atthe outer surface of the reagent layer. If the layer is in directcontact with blood, i.e., when no intervening separation layer has beendeposited, red and white blood cells, fat and proteins present in thesample can interact with the reagent layer and interfere with themeasurement of the amount of analyte in the sample.

In contrast, in the present invention, the integrated reagent/bloodseparation layer is non-conductive, and thus is not a part of theelectrode either structurally or functionally. Change transfer does notoccur unless electroactive species pass through the openings/pores ofthe integrated reagent/blood separation layer to reach the underlyingconductive element. Thus, the integrated reagent/blood separation layerprovides a barrier to the passage of interferents such as cells andmacromolecules to the conductive element resulting in a device withsuperior properties that is simpler to make.

In achieving this result, it is particularly desirable that theintegrated reagent/blood separation layer be deposited in such a waythat no portion of the conductive element 16 be directly exposed to thesample when it is placed in the sample application region. Themethodology described above, in which an insulating layer with aperturesproviding access to the conductive elements 14′ and 16 is utilized isparticularly suited for achieving this result. Thus, as shown in FIGS.9A-C, this methodology allows the formation of the test strip in onlythree steps. In the first step (FIG. 9A), two conductive elements 14′and 16 and associated leads and contacts are deposited on a substrate.In a second step (FIG. 9B), a layer of insulating material is depositedover the conductive elements. The insulating material has two apertures94 and 96, one in alignment with each of the conductive elements 14′ and16. In the third step, (FIG. 9C), the integrated reagent/bloodseparation layer 17 is deposited over the aperture 96. By making thedeposited layer 17 larger in dimensions than the aperture 96, thereagent layer completely covers the underlying conductive element andthat it is not exposed directly to the sample, thereby providingeffective blood separation.

The complete coverage of conductive element 16 also addresses anothersources of error which can occur as a result of electrochemicaloxidation or small molecules such as ascorbic acid, uric acid andacetaminophen which may be present in the sample. When present, theoxidation of these molecules at the surface of the electrode leads tospuriously elevated current levels, and thus an inaccurate measurementof the desired analyte, e.g. glucose. The integrated reagent/bloodseparation layer of invention will not generally exclude thesemolecules, since they are small compared to the pore sizes observed.However, by including a pH buffer in the integrated reagent/bloodseparation layer one can shift the local pH at the electrode surface toa level where electrochemical potential of these species is higher.Thus, for example, the use of an integrated reagent/blood separationlayer in which the pH is buffered to a level of around pH 5 willsubstantially reduce the impact of these interferents. To maximize theeffectiveness of this buffering, however, the entire conductive elementmust be covered, since even a relatively small region of exposed (notbuffered) electrode surface can result in a large interference current.

Not only do the test strips of the invention provide performancebenefits resulting from the separation of the conductive element fromthe blood sample, the test strips of the invention are also resistant toother sources of error. For example, during the period of a test,reagents may diffuse laterally away from the original deposit. If thereagent layer is deposited directly on the conductive element, thesereagents will continue to contribute to the measured signal. Anyvariations in convective diffusion from test to test (for example as aresult of differences in temperature or differences in the handling ofthe instrument) will therefore be manifested as irreproducibility in thesignal. If the reagent layer overlaps the insulation print, however,lateral diffusion away from the aperture will not contribute to thesignal and therefore will not give rise to variations in the signal.

In addition to providing a test strip with beneficial properties, themethodology outlines in FIGS. 9A-C offers several advantages from amanufacturing perspective. First, if the reagent layer is printeddirectly onto the conductive element, the “active area” is defined bythe area of the reagent layer. The precision of the test is thereforedetermined by the precision with which the reagent layer can be printed.In contrast, by first depositing an apertured insulation layer definingthe region of the contact between the reagent layer and the underlyingconductive element, the active area is defined by the size of theaperture in the insulation layer. Since insulation layers are typicallyprinted using a finer screen, much better edge definition, and thusgreater device precision can be achieved. Thus, neither the area ofconductive element 16 nor of the integrated reagent/blood separationlayer are critical to the performance characteristics of the finishedtest strip. The conductive elements and the integrated reagent/bloodseparation layer may therefore be applied using techniques which provideless precision than can be employed in other processes.

It will be appreciated by persons skilled in the art that, while bothconductive elements must be accessible to electroactive species in asample disposed in the sample application region, the important functionof the insulation mask is to provide an aperture defining the contactregion between conductive element 16 and the integrated reagent/bloodseparation layer 17. Thus, in the limiting case, it is only necessary toform one aperture in the insulation layer. The second conductive elementcan be exposed along an edge of the insulation layer, or may be locatedon a facing surface in a folded electrode structure.

The substrate 10 used in making the test strips of the invention can beany non-conducting, dimensionally stable material suitable for insertioninto a glucose test meter. Suitable materials include polyester films,for example a 330 micron polyester film, and other insulating substratematerials such as polyvinyl chloride (PVC) and polycarbonate.

The conductive elements and associated leads and contacts can be formedfrom essentially any conductive material including silver, Ag/AgCl,gold, or platinum/carbon, and need not all be formed from the samematerial. The conductive element 16 is preferably formed from conductivecarbon. Preferred conductive carbon are ERCON ERC1, ERCON ERC2 andAcheson Carbon Electrodag 423. Carbon with these specifications isavailable from Ercon Inc. (Waltham, Mass., USA), or Acheson Colloids,(Princes Rock, Plymouth, England). The conductive element 16 makescontact with working electrode track 15, and is close to, but notcontacting conductive element 14′ disposed as the end of referenceelectrode track 14.

The insulating layer 18 can be formed from polyester-based printabledielectric materials such as ERCON R488-B(HV)-B2 Blue. The top cover 23is suitably formed from a polyester strip or a “hot melt” coatedplastic.

The test strips of the present invention do not require the formation ofa discrete exit port to permit air to escape from the device as sampleenters the electrode chamber but instead uses a distributed exit alongall of the edges of the mesh. As the sample fluid wicks along the mesh,air seeps out of the edges of the mesh all around the device underneaththe top layer. The sample fluid does not seep out because the insulationlayer imparts significant hydrophobicity to that part of the mesh. Theliquid sample therefore remains in the central hydrophilic region.

The key to the performance achieved using the present invention is inthe nature of the integrated reagent/blood separation layer 17. Thislayer can be formed from a mixture containing a filler which has bothhydrophobic and hydrophilic surface regions, and in the case of aglucose test strip, an enzyme which can oxidize glucose, and a mediatorwhich can transfer electrons from the enzyme to the underlyingconductive element layer 16. This layer is suitably formed byformulating an ink which contains the filler, the enzyme and themediator in a suitable carrier and using this ink to print the layer 17onto the device.

A preferred filler for use in the layer 17 is silica. Silica isavailable in a variety of grades and with a variety of surfacemodividations. While all silica compounds tested resulted in a productwhich could measure glucose under some conditions, the superiorperformance characteristics of glucose test strip of the invention areobtained when a silica having a surface modification to render itpartially hydrophobic is used. Materials of this type include Cab-O-SilTS610, a silica which is modified by partial surface treatment withmethyl dichlorosilane; Cab-o-Sil 530, a silica which is modified by fullsurface treatment with hexamethyl disilazane; Spherisorb C4 silica,which is surface modified with 4 carbon chains; and other similarlymodified silicas; or combinations thereof. Silica with a surfacemodification which is too hydrophobic should be avoided. For example, ithas been observed that C18-modified silica is too hydrophobic to form aprintable ink.

During the process of manufacturing the ink of the invention, theparticles are broken down by homogenization to expose hydrophilic innerprotions of the silica particles. The actual particles present in theink therefore have both hydrophilic and hydrophobic regions. Thehydrophilic regions form hydrogen bonds which each other and with water.

When this material is formulated into an ink as described below inExample 1, and screen printed onto the conductive element 16, the dualnature of the material causes it two form layers of two-dimensionalnetworks which take from as a kind of honeycomb which is visible uponmicroscopic examination. On rehydration; this layer does not break up,but swells to form a gelled reaction zone in the vicinity of theunderlying conductive element 16. Reactants such as enzyme, mediator andglucose move freely within this zone, but interfering species such asred blood cells containing oxygenated hemoglobin are excluded. Thisresults in a device in which the amount of current generated in responseto a given amount of glucose varies by less than 10 percent over ahematocrit range of 40 to 60%, and which is thus substantiallyinsensitive to the hematocrit of the sample, and in fact performssubstantially the same in blood as in a cell-free control solution.(FIGS. 3A-C, FIG. 4 and FIG. 5A-5C)

Furthermore, the gelled reaction zone presents a greater barrier toentry of blood analytes such as glucose which makes the devicediffusion, rather than kinetically limited. This leads to a device inwhich the measured current varies by less than 10 percent over atemperature range from 20° C. to 37° C. and which is thus essentiallytemperature independent. (FIGS. 6A and 6B)

When making a glucose test strip, the integrated reagent/bloodseparation layer is advantageously formed from an aqueous compositioncontaining 2 to 10% by weight, preferably 4 to 10% and more preferablyabout 4.5% of a binder such as hydroxyethylcellulose or mixtures ofhydroxyethylcellulose with alginate or other thickeners; 3 to 10% byweight, preferably 3 to 5% and more preerably about 4% silica; 8 to 20%by weight, preferably 14 to 18% and more preferably about 16% of amediator such as ferricyanide; and 0.4 to 2% by weight, preferably 1 to2% and more preferably about 1.6% of an enzyme such as glucose oxidase,assuming a specific activity of about 250 units/mg, or about 1000 to5000 units per gram of ink formulation.

The integrated reagent/blood separation layer may also includeadditional ingredients without departing from the scope of theinvention. For example, the nonconducting layer may include an antifoam.In addition, the nonconducting layer may be formulated with a bufferingagent to control the pH of the reaction zone. The pH may be maintainedat a level within the range from about pH 3 to pH 10. In one embodimentof the invention, it is of particular utility to maintain the pH of thedevice at a level above 8 because at this pH oxygen bound to hemoglobinis not released. Further, at this pH, the reaction rate of glucoseoxidase with oxygen is very low. Thus, selection of an appropriate pHcan further stabilize the performance of the test strip against theeffects of varying hematocrit. In an alternative embodiment of theinvention, maintaining a low pH (below pH 5.5, the optimium pH forreaction of glucose oxidase with oxygen) may be preferred. For example,maintaining a pH of around pH 5 is better if the primary concern is theelimination of electrochemical interferences arising from oxidation ofinterfering substances such as ascorbic acid, uric acid oracetaminophen, since these compounds are more difficult to oxidize atlower pH.

While a preferred embodiment of the invention is a glucose test strip asdescribed above, the test strips of the invention are not limited to thedetection of glucose. For example, a fructosamine test strip couldinclude two layers disposed over the conductive element. The first,lower layer is formed from an ink comprising a carbonate buffer (pH>10)in a silica mix substantially as described in Example 7 but withoutenzyme, mediator or citrate buffer. The second, upper layer is formedfrom an ink further comprising an oxidant such as ferricyanide.

FIG. 7 shows an alternative embodiment of the invention. In thisembodiment, a second non-conductive layer 71 is disposed over theintegrated reagent/blood separation layer 17. This layer is formed froma composition which is identical to the first integrated reagent/bloodseparation layer except that the enzyme or both the enzyme and themediator are omitted. This layer further isolates the conductive element16 from contact with oxygen-carrying red blood cells, thus reducing theeffects of oxygen. Furthermore, to the extent that enzyme may tend todiffuse away from the surface of the electrode during the course of themeasurement such a layer containing mediator can provide an increasedregion in which it will have mediator available for the transfer ofelectrons.

EXAMPLE 1

A non-conducting formulation for preparation of the integratedreagent/blood separation layer 17 was made as follows. 100 ml of 20 mMaqueous trisodium citrate was adjusted to pH 6 by the addition of 0.1 Mcitric acid. To this 6 g of hydroxyethyl cellulose (HEC) was added andmixed by homogenization. The mixture was allowed to stand overnight toallow air bubbles to disperse and then used as a stock solution for theformulation of the coating composition.

2 grams Cab-o-Sil TS610 silica and 0.1 grams of Dow Corning antifoamcompound was gradually added by hand to 50 grams of the HEC solutionuntil about ⅘ of the total amount had been added. The remainder wasadded with mixing by homogenization. The mixture was then cooled for tenminutes in a refrigerator. 8 g of potassium hexacyanoferrate (III) wasthen added and mixed until completely dissolved. Finally, 0.8 g ofglucose oxidase enzyme preparation (250 Untis/mg) was added and thenthoroughly mixed into the solution. The resulting formulation was readyfor printing, or could be stored with refrigeration.

EXAMPLE 2

To prepare glucose test strips using the ink formulation of Example 1, aseries of patterns are used to screen print layers onto a 330 micronpolyester substrate (Melinex 329). The first step is the printing ofcarbon pads. An array of 10×50 pads of carbon is formed on the surfaceof the polyester substrate by printing with EC2 carbon. (Ercon) Theprinted substrate is then passed through a heated dryer, and optionallycured at elevated temperature (e.g. 70° C.) for a period of 1 to 3weeks.

Next, an array of silver/silver chloride connecting tracks and contactsis printed onto the substrate using ERCON R-414 (DPM-68) 1.25bioelectrode sensor coating material and dried. One working track whichmakes contact with the carbon pad and one refernce track is printed foreach carbon pad in the array.

A dielectric layer is then printed using ERCON R488-B(HV)-B2 Blue anddried. The dielectric layer is printed in a pattern which coverssubstantially all of each device, leaving only the contacts, the tip ofthe reference electrode and the carbon pads uncovered.

On top of the dielectric layer the ink of Example 1 is used to form aintegrated reagent/blood separation layer overlaid on top of eachconductive carbon pad.

Polyester mesh strips (Scryncl PET230 HC) arethen laid down across thesubstrate in lines, covering the reactions areas exposed by the windowsin the dielectric. An 5 mm wide polyester strip (50 microns thick) isthen applied over the top of the mesh strips, and the edges of theelectrodes are heat sealed. Finally, the substrate is cut up to provide50 individual electrodes, for example having a size of 5.5 mm wide and30 mm long.

EXAMPLE 3

Test strips manufactured using the ink formulation of Example 1 in themanner described in Example 2 were placed in a test meter with anapplied voltage of 500 mV and used to test blood samples having varyingglucose concentrations and hematocrits ranging from 40% to 60%. FIGS.3A-3C show the current measured 25 seconds after applying the voltage asa function of the glucose concentration, and FIG. 4 plots the slope ofthe glucose response as a function of hematocrit. As can be seen, theindicators produce highly reproducible current levels which areessentially independent of hematocrit.

EXAMPLE 4

Glucose test strips in accordance with the invention were made inaccordance with Example 2, except the non-conductive layer was formedwith 7 g Spherisorb C4 and 1 g Cab-o-Sil TS610. This formulation waslaid down on three different types of carbon-containing conductiveelements as follows:

A: Ercon EC1

B: Ercon EC2

C: Ercon EC2 on top of Acheson Carbon, Electrodag 423 SS.

These test strips were used to measure varying levels of glucose ineither a control solution (One Touch Control Solution, Lifescan Inc.)containing glucose in an inert solution or in blood at an appliedvoltage of 425 mV. The current observed 25 seconds after the voltage wasapplied was measured. FIGS. 5A-5C show the results obtained for thethree formulations, A, B, and C, respectively. In all cases, the slopeof the line showing the response of the meter to different glucoseconcentrations was essentially the same whether the measurements weremade in blood or the control solution. Thus, this further demonstratesthe independence of the test strips of the invention from the oxygencontent and hematocrit of the sample, as well as the ability to suevarious materials as the conductive element.

EXAMPLE 5

Test strips prepared in accordance with Example 2 were tested at twodifferent sample temperatures, namely 37° C. and 20° C. using an appliedvoltage of 425 mV. FIGS. 6A and 6B show the current measured 25 secondsafter applying the voltage as a function of glucose concentration. Ascan be seen, the slopes of the two lines are essentially identical(0.1068 at 20° C. versus 0.1009 at 37° C.), thus demonstrating that thetest stips provide essentially temperature-independent behavior over atemperature range from ambient to physiological temperatures.

EXAMPLE 6

The current transient was measured for a test strip prepared inaccordance with Example 2 and for a commercial test strip made with acarbon-containing ink. The results are shown in FIGS. 8A and 8B. Asshown, the test strip of the invention (FIG. 8A) provides a very flattransient which maintains more than 50% of the peak current for a periodof more than 25 seconds after the initial response from the test strip.In contrast, the carbon-based electrode exhibited an almost immediatedecay in the current, having lost 50% of the peak current in a period ofthe first 1 to 2 seconds after the initial response from the test strip.This makes timing of the measurement difficult if peak current valuesare to be captured, or reduces the dynamic range of the meter if currentmust be measured after substantial decay has occurred. Thus, the teststrips of the invention are advantageous in that the current generatedin response to a given amount of glucose decays by less than 50% in the5 seconds following peak current generation.

EXAMPLE 7

An ink for printing glucose test strips in accordance with the inventionwas formulated as follows:

67.8 g 20 mM Citrate buffer, pH 6

0.68 g Polyvinyl alcohol (MW 85,000-146,000, 88% hydrolysed)

0.68 g of Polyvinyl pyrrolidone-vinyl acetate

0.42 g of Dow Corning DC1500 antifoam

3.4 g of hydroxyethyl cellulose (Natrosol 250G, Hercules)

5.5 g of surface modified silica (Cabo-Sil TS610, Cabot)

1.5 g glucose oxidase

20.0 g Potassium Ferricyanide.

EXAMPLE 8

FIGS. 10A-I shows the stepwise preparation of a test strip in accordancewith the invention. As is apparent from a comparison of this test stripand the strip of FIG. 1, the precise arrangement of the electrodes onthe strip is not critical. Further, different materials may be used infabricating the strip.

The first step in fabricating the test strip is the deposition of silvertracks 101, 102 of substrate 100. A preferred substrate is a 500 micronthick polyester film sold under the tradename Valox™. The silverelectrodes can be formed by screen printing using an ink compositionformulated as in Example 2.

After deposition of the silver electrodes, a second electrode print iscarried out to form carbon conductive elements 103, 104 and 105 as shownin FIG. 10B. Conductive element 103 is formed in contact with silvertrack 101 and will form the working electrode in the finished teststrip. Carbon pads 104 and 105 connect electrically to the ends ofsilver tracks 101 and 102 and provide connection between the strip and atest meter. The carbon conductive elements can be formed by screenprinting with a conductive carbon ink formulation such as thosedescribed in the previous examples.

The next step in the manufacturing process is the deposition of aninsulation layer 106 for example by screen printing an insulation ink,for example the dielectric ink of Example 2. (FIG. 10C) As shown, theinsulation layer contains three windows 107, 108 and 109. Window 108 isaligned with the end of the carbon conductive element 103. Window 107 isaligned with the end of silver track 102 to provide access to therefrence electrode. The third window, 109, is provided to permit passageof insulation material from the second 9insulation coating through themesh layer, but is not required.

FIG. 10D shows the next step in the process, which is the formation ofan integrated reagent/blood separation layer 110. This layer isdoposited over window 108 and extends over the insulation layer 106along all sides of the window 108. A suitable formulation for printinglayer 110 has the following composition to provide an integratedreagent/blood separation layer with a buffered pH of about 6: ComponentAmount Analar Water 3 L Tri-sodium Citrate 15.75 g Nat 250 G 150 gCitric Acid 6.3 g Poly Vinyl Alcohol 30 g DC 1500 Defoamer 15 ml Cabosil225 g Glucose Oxidase 48 g Potassium Hex/60299 660 g PVPVA 30 g

After the integrated reagent/blood separation layer 110 is formed, alayer of mesh 111 is deposit over the sample collection region of thetest strip. (FIG. 10E) The mesh 111 is preferably a nylon mesh which hasbeen pretreated with acetone and Fluorad FC 170C surfactant to renderthe mesh hydrophilic. The purpose of the mesh 111 is the transport ofthe liquid sample evenly through the area between the working andreference electrodes.

A second insulation print 112 is then carried out using a slightly moreflexible insulation ink (ERCON Insulayer 820202) to define the samplecollection region. (FIG. 10F). A tape cover 113 is then applied over thetop of the test strip as described above in Example 2 to form a finishedtest strip. (FIG. 10G).

1-23. (canceled)
 1. A disposable test strip for use in a test meterwhich receives a disposable test strip and a sample of blood andperforms an electrochemical analysis of the amount of a blood analyte inthe sample, comprising: (a) a substrate; (b) a first conductive elementdisposed on the substrate; (c) a second conductive element disposed onthe substrate in sufficient proximity to the first conductive element toallow the completion of an electrical circuit between the first andsecond conductive elements when a sample of blood is placed on the teststrip; (d) a non-conductive integrated reagent/blood separation layerdisposed over the first conductive element, the integrated reagent/bloodseparation layer comprising reagents for the electrochemical detectionof the analyte dispersed ina non-conductive matrix effective to excludeblood cells from the surface of the first conductive element whilepermitting access to the first conductive element by solubleelectroactive species; (e) contacts for making an electrical connectionbetween the first and second conductive elements and the test meter; and(f) an insulation layer disposed over at least the first conductiveelement, the insulation layer having a first aperture therein alignedwith the first conductive element, the insulation layer having aperipheral portion adjacent to a surrounding area of the first aperture,wherein the non-conductive integrated reagent/blood separation layer isformed covering the entire first aperture and the peripheral portion. 2.The disposable test strip of claim 1, wherein a sufficient amount of theperipheral portion is covered by the non-conductive integratedreagent/blood separation layer to reduce variation in a measured signalas a result of lateral diffusion away from the first aperture
 3. Thetest strip of claim 1, wherein the integrated reagent/blood separationlayer comprises an enzyme for oxidation of glucose and a redox mediatoreffective to transfer electrons from the enzyme to the first conductiveelement.
 4. The test strip of claim 3, wherein the matrix comprisessilica having both hydrophobic and hydrophilic surfaces.
 5. The teststrip of claims 1, wherein the first and second conductive elementscomprise conductive carbon.
 6. The test strip according to claim 3,wherein the enzyme is glucose oxidase.
 7. The test strip according toclaim 3, wherein the redox mediator is ferricyanide.
 9. The test stripaccording to claim 4, wherein the integrated reagent/blood separationlayer is formed from an aqueous composition comprising 2 to 10% byweight of a binder; 3 to 10% by weight of silica; 8 to 20% by weight ofthe redox mediator; and 1000 to 5000 units of the enzyme per gram of theaqueous composition.
 10. The test strip according to claim 9, whereinthe enzyme is glucose oxidase.
 11. The test strip according to claim 9,wherein the redox mediator is ferricyanide.